A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one (3-carotene, and one or two cytochrome b-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem H reaction center. These results strongly suggest that the site of primary charge separation in photosystemIH is located on the heterodimer composed of D-1 and D-2 subunits.It has been well established that the photosystem II reaction center of oxygenic photosynthetic organisms is contained in a chlorophyll-protein complex consisting of the following six polypeptide subunits: 47-and There is a controversy concerning the localization of the site of primary charge separation in the photosystem II reaction center complex (5-10). Some evidence supports the proposal that the 47-kDa subunit is the site of primary photochemistry (5-8). However, the amino acid sequence homology between the D-1 and D-2 subunits and the L and M subunits of reaction center from purple photosynthetic bacteria has led to a proposal (9, 10) that the D-1 and D-2 polypeptides play a role in the photosystem II reaction center similar to that of L and M subunits in the bacterial reaction center whose structure has been determined by x-ray crystallographic analysis (11).In this paper we provide experimental evidence supporting the latter proposition. We have succeeded in isolating a pigment-protein complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 that is capable of reversible photochemical accumulation of reduced pheophytin. A preliminary account of this work has been presented (12). MATERIALS AND METHODSThe membrane preparation of grana thylakoids (Triton/ photosystem II particles) was prepared from spinach as described by Kuwabara and Murata (13).Pigments and quinones were extracted with 80% (vol/vol) acetone and quantitatively determined by HPLC using a reverse-phase column (ZORBAX-ODS). Following the procedure described (14), methanol/water, 49:1 (vol/vol) and methanol/isopropanol, 3:1 (vol/vol) were used for chromatographic development, and the elution of components was monitored by the absorption either at 440 nm (chlorophylls and carotenoids) or at 255 nm (pheophytin a and plastoquinone-9). The spectrophotometry of the components was based on the absorption coefficients described by Eskins et al. (14) for chlorophylls and carotenoids, by Vernon (15) for pheophytins, and by Barr and Crane (16) for plastoquinones. The amounts of cytochrome b-559 with different midpotentials were determined by the method of Hind and Nakatani (17) using a difference millimolar absorption coefficient (559-570 nm) of 15 (18). NaDodSO4/polyacrylamide gel electrophoresis was carrie...

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